**1. Introduction**

The transfer of extracellular vesicles (EVs) has emerged in the last two decades as a novel mechanism for intercellular communication. Nomenclature of EVs has by now been agreed to be based on biogenesis pathway. Therefore, EVs budding from plasma membranes are termed ectosomes or microvesicles, while exosomes are formed via the endosomal compartment within multivesicular bodies (MVB) which then are released by their fusion with the plasma membrane. Finally, when blebbing from apoptotic cells, the term apoptotic bodies has been retained [1]. Biomarkers to differentiate these EVs are still questionable, and in general, size is used to differentiate exosomes from ectosomes, while apoptotic bodies are considered to present phosphatidylserine on the outside and thus can be stained by annexin V. Exosomes (40–150 nm in diameter) are produced by formation of endosomal intraluminal vesicles (ILVs) in multivesicular bodies (MVBs) and are secreted by fusion of these vesicles with the plasma membrane [2, 3] (**Figure 1**).

**Figure 1.** *Biogenesis of EVs.*

Since they have been known to mediate directional intercellular transfer of their content, typically proteins, mRNAs, microRNAs (miRNAs) and a variety of noncoding RNAs [4], several studies were oriented towards their application as drugs *per se* or as therapeutic tool for drug delivery [5]. Their membrane is strongly enriched in sphingomyelin and cholesterol, which contributes to their unique buoyancy coefficient and enables practical isolation from other particles of cellular origin using differential centrifugation methods. Another discernible feature of exosome membranes is that it reflects the composition of the MVB membrane and has a high density of endosomal membrane proteins, such as proteins involved in MVB biogenesis (Alix and TSG101), membrane transport, in particular the components of the endosomal sorting complex required for transport (ESCRT) and most prominently tetraspanin proteins (CD9, CD37, CD53, CD63, CD81, CD82 and CD151) [6, 7]. Some tetraspanins such as CD9, CD81 and CD151 are more broadly expressed, while others are restricted to specific EV subsets [8, 9]. The overexpression of tetraspanins, as shown for CD9, can in several production cell lines enhance the exosome production and, in addition, cause a reduced overall size of the vesicles [10].

Tetraspanins play an important role in several physiological processes [11] and have been discovered to cooperate in states of health and disease in signal transduction, cellular activation, polarization, motility, adhesion, tissue differentiation, angiogenesis, tumorigenesis and metastasis [12–14], both by regulating cellular interactions as cell-membrane bound molecules and indirectly through exosomes. They are involved in each step of the metastatic cascade due to their ability to interact with cell surface receptors, adhesion molecules, matrix-remodeling proteases and signaling molecules. In this pathological state, they hence regulate cell proliferation, participate in epithelial-mesenchymal transition, modulate integrin-mediated cell adhesion and mediate the invasion through modulation of angiogenesis, tumorendothelial cell interactions and regulation of cancer cell migration through the regulation of tumor microenvironment, as well as direct influence on extracellular matrix [15]. In this chapter, we introduce the characteristics of EVs and the engineering approaches aimed at their surface proteins to achieve tissue-specific targeting.

## **2. Structure of the tetraspanin proteins**

The rod-shaped structure of a tetraspanin consists of four transmembrane helices that connect the two extracellular loops [16] (**Figure 2**). The short extracellular loop (SEL), which has not yet been reported to contain any element of a

**107**

the lipid rafts [26].

tetraspanin [17].

**Figure 2.**

*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting*

particular secondary structure, is of 12–31 amino acids in length. In the studies oriented towards tetraspanin engineering, the EC1 domain has until now been less addressed due to its lower degree of structural organization. Its replacement for a strand of glycine-serine residues has led to an unusual cell surface expression and a distribution dissimilar to the wild-type parental molecule, which may suggest the importance of this structural subunit for the stability of the membrane-bound

*Organization of tetraspanin CD81: 4 transmembrane helices (TM) span 2 extracellular loops. Positions of cysteine residues are indicated with yellow dots. Crystal structure of EC2 domain is presented as a cartoon diagram of the dimeric form (PDB: 1G8Q ); the conserved three-helix bundle is black and the variable domains C and D are surfaced in blue or green for each protomer. The figure was prepared using PyMOL* 

The large extracellular loop (LEL) or extracellular domain 2 (EC2) has on the other hand been well characterized. The crystal structure of CD81 [18] demonstrated the four invariant cysteine resides within EC2 to form the 2 disulfide bridges, as a hallmark stability feature of the conserved tetraspanin fold [8, 19] (**Figure 2**). Moreover, the topology of several other tetraspanins was predicted by molecular modeling studies in the absence of an available crystal structure, using the CD81 EC2 structure as a template: these were the tetraspanins CD37, CD53, CD82 and CD151 [20, 21]. It has been early established that binding of CD81-specific antibodies depends on the formation of the disulfide bonds [22]. One class of tetraspanins harbors the EC2 composed of five α-helical elements, forming stalk and head elements of a mushroom-like structure, and the second class of tetraspanins is postulated to contain an additional helical element, stabilized with another pair of cysteine residues. The EC2 fold structure is evolutionarily conserved across species for any particular tetraspanin in spite of a high degree of variability at the amino acid level [23], with a subdomain consisting of a three-helix-bundle fold and a second subdo-

main variable in size among members of the tetraspanin family [18, 20].

The presence of an energetically unfavorable hydrophobic patch and the dimeric form of CD81 LEL in a crystal hinted at the high likelihood of tetraspanin assembly into dimers or multimers in the cell membrane. As shown later, clustering of tetraspanins leads to the organization of tetraspanin-associated proteins in a tetraspanin web or tetraspanin-enriched microdomain (TEM) [24, 25]. Biologically, such domains induce partitioning of the complexes into lipid rafts and clustering of

Apart from the hydrophobic interactions of the extracellular domains, the membrane-proximal cysteines residing in transmembrane domains contribute to the dimer formation and stability of tetraspanins. For the CD9 tetraspanin, it has been demonstrated that these residues are positioned close to the dimerization interface and influence homotypic and heterotypic tetraspanin association depending on their reversible palmitoylation status [27]. Similarly, the mutation of N-terminal

*DOI: http://dx.doi.org/10.5772/intechopen.83537*

*(PyMOL molecular graphics system, version 1.3 Schrödinger, LLC).*

*Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting DOI: http://dx.doi.org/10.5772/intechopen.83537*

**Figure 2.**

*Current Topics in Biochemical Engineering*

**Figure 1.** *Biogenesis of EVs.*

Since they have been known to mediate directional intercellular transfer of their content, typically proteins, mRNAs, microRNAs (miRNAs) and a variety of noncoding RNAs [4], several studies were oriented towards their application as drugs *per se* or as therapeutic tool for drug delivery [5]. Their membrane is strongly enriched in sphingomyelin and cholesterol, which contributes to their unique buoyancy coefficient and enables practical isolation from other particles of cellular origin using differential centrifugation methods. Another discernible feature of exosome membranes is that it reflects the composition of the MVB membrane and has a high density of endosomal membrane proteins, such as proteins involved in MVB biogenesis (Alix and TSG101), membrane transport, in particular the components of the endosomal sorting complex required for transport (ESCRT) and most prominently tetraspanin proteins (CD9, CD37, CD53, CD63, CD81, CD82 and CD151) [6, 7]. Some tetraspanins such as CD9, CD81 and CD151 are more broadly expressed, while others are restricted to specific EV subsets [8, 9]. The overexpression of tetraspanins, as shown for CD9, can in several production cell lines enhance the exosome production

Tetraspanins play an important role in several physiological processes [11] and have been discovered to cooperate in states of health and disease in signal transduction, cellular activation, polarization, motility, adhesion, tissue differentiation, angiogenesis, tumorigenesis and metastasis [12–14], both by regulating cellular interactions as cell-membrane bound molecules and indirectly through exosomes. They are involved in each step of the metastatic cascade due to their ability to interact with cell surface receptors, adhesion molecules, matrix-remodeling proteases and signaling molecules. In this pathological state, they hence regulate cell proliferation, participate in epithelial-mesenchymal transition, modulate integrin-mediated cell adhesion and mediate the invasion through modulation of angiogenesis, tumorendothelial cell interactions and regulation of cancer cell migration through the regulation of tumor microenvironment, as well as direct influence on extracellular matrix [15]. In this chapter, we introduce the characteristics of EVs and the engineering approaches aimed at their surface proteins to achieve tissue-specific targeting.

The rod-shaped structure of a tetraspanin consists of four transmembrane helices that connect the two extracellular loops [16] (**Figure 2**). The short extracellular loop (SEL), which has not yet been reported to contain any element of a

and, in addition, cause a reduced overall size of the vesicles [10].

**2. Structure of the tetraspanin proteins**

**106**

*Organization of tetraspanin CD81: 4 transmembrane helices (TM) span 2 extracellular loops. Positions of cysteine residues are indicated with yellow dots. Crystal structure of EC2 domain is presented as a cartoon diagram of the dimeric form (PDB: 1G8Q ); the conserved three-helix bundle is black and the variable domains C and D are surfaced in blue or green for each protomer. The figure was prepared using PyMOL (PyMOL molecular graphics system, version 1.3 Schrödinger, LLC).*

particular secondary structure, is of 12–31 amino acids in length. In the studies oriented towards tetraspanin engineering, the EC1 domain has until now been less addressed due to its lower degree of structural organization. Its replacement for a strand of glycine-serine residues has led to an unusual cell surface expression and a distribution dissimilar to the wild-type parental molecule, which may suggest the importance of this structural subunit for the stability of the membrane-bound tetraspanin [17].

The large extracellular loop (LEL) or extracellular domain 2 (EC2) has on the other hand been well characterized. The crystal structure of CD81 [18] demonstrated the four invariant cysteine resides within EC2 to form the 2 disulfide bridges, as a hallmark stability feature of the conserved tetraspanin fold [8, 19] (**Figure 2**). Moreover, the topology of several other tetraspanins was predicted by molecular modeling studies in the absence of an available crystal structure, using the CD81 EC2 structure as a template: these were the tetraspanins CD37, CD53, CD82 and CD151 [20, 21]. It has been early established that binding of CD81-specific antibodies depends on the formation of the disulfide bonds [22]. One class of tetraspanins harbors the EC2 composed of five α-helical elements, forming stalk and head elements of a mushroom-like structure, and the second class of tetraspanins is postulated to contain an additional helical element, stabilized with another pair of cysteine residues. The EC2 fold structure is evolutionarily conserved across species for any particular tetraspanin in spite of a high degree of variability at the amino acid level [23], with a subdomain consisting of a three-helix-bundle fold and a second subdomain variable in size among members of the tetraspanin family [18, 20].

The presence of an energetically unfavorable hydrophobic patch and the dimeric form of CD81 LEL in a crystal hinted at the high likelihood of tetraspanin assembly into dimers or multimers in the cell membrane. As shown later, clustering of tetraspanins leads to the organization of tetraspanin-associated proteins in a tetraspanin web or tetraspanin-enriched microdomain (TEM) [24, 25]. Biologically, such domains induce partitioning of the complexes into lipid rafts and clustering of the lipid rafts [26].

Apart from the hydrophobic interactions of the extracellular domains, the membrane-proximal cysteines residing in transmembrane domains contribute to the dimer formation and stability of tetraspanins. For the CD9 tetraspanin, it has been demonstrated that these residues are positioned close to the dimerization interface and influence homotypic and heterotypic tetraspanin association depending on their reversible palmitoylation status [27]. Similarly, the mutation of N-terminal

and C-terminal transmembrane cysteines to serine eliminated palmitoylation of CD151, which turned out to be deleterious for the assembly with other cell surface proteins, including tetraspanins CD9 and CD63, their organization to TEMs and subsequently their subcellular distribution and cell morphology [28]. At the same time, it had minimal influence on the density of tetraspanin protein complexes and was dispensable for CD151-α3β1 integrin association. Depalmitoylation of CD81 did not impact its surface expression and stability, but rendered it less available for contact with its natural interaction partner CD9 and the relevant epitopes less accessible for binding of structurally dependent antibodies [29].

The four transmembrane helices of tetraspanin proteins form two largely separated pairs of antiparallel helices: one pair comprises TM1/TM2 and the other TM3/TM4. The two pairs of helices only converge close to the cytoplasmic side of the membrane through contacts between TM2 and TM3. In the recently solved crystal structure of full-length CD81, this cone-like structure has been shown to harbor a binding pocket for cholesterol [30], and mutations within transmembrane domains in certain tetraspanins have been connected with pathological states [31]. The short cytoplasmic tails show no obvious functional significance in signaling processes, suggesting that their signaling competence relies on association with other molecules [32]; nevertheless, its mutation can lead to different assembly with association partners as shown for CD9 [33].
